The invention relates to novel compounds that are effective anti-cancer agents and potent inhibitors of tubulin polymerization.
Cancer is a major disease that continues as one of the leading causes of death at any age. In the United States alone, it is estimated that more than a half a million Americans will die annually of cancer. Currently, radiotherapy and chemotherapy are two important methods used in the treatment of cancer.
Considerable efforts are underway to develop new chemotherapeutic agents for more potent and specific anti-cancer therapy, presenting effective and efficient cytotoxicity against tumor cells, with minimal interference with normal cell function. Accordingly, there is an urgent need for the development and analysis of novel, effective anti-cancer agents.
Cellular proliferation, for example, in cancer, occurs as a result of cell division, or mitosis. Microtubules play a pivotal role in mitotic spindle assembly and cell division. These cytoskeletal elements are formed by the self-association of the xcex1xcex2 tubulin heterodimers.
Recently, the structure of the xcex1xcex2 tubulin dimer was resolved by electron crystallography of zinc-induced tubulin sheets. According to the reported atomic model, each 46xc3x9740xc3x9765 xc3x85 tubulin monomer is made up of a 205 amino acid N-terminal GTP/GDP binding domain with a Rossman fold topology typical for nucleotide-binding proteins, a 180 amino acid intermediate domain comprised of a mixed xcex2 sheet and five helices which contain the taxol binding site, and a predominantly helical C-terminal domain implicated in binding of microtubule-associated protein (MAP) and motor proteins.
As disclosed in U.S. Pat. No. 6,258,841 B1, tubulin has a binding pocket in a region within the intermediate domain of tubulin, located between the GDP/GTP binding site and the taxol binding site. The approximate dimensions of the binding pocket are 6 xc3x85xc3x9722 xc3x85xc3x977 xc3x85. The pocket, referred to as the COBRA binding pocket, has an abundance of leucine residues (7 leucine and 2 isoleucine) providing a highly hydrophobic binding environment. It is characterized by a narrow cavity with elongated dimensions, suitable for accommodating an aliphatic chain of up to about 12 carbons.
Compounds that bind with the COBRA binding pocket can interfere with tubulin polymerization and can provide novel agents for the treatment of cancer.
The compounds of the invention bind to tubulin causing tubulin depolymerization and inhibiting tubulin polymerization. The tubulin binding compounds of the invention are therapeutically effective to inhibit cellular proliferation, for example, as effective anti-cancer agents. The compounds are cytotoxic against tumor cells such as leukemia cells. The compounds are novel furan, thiophene, thiazole, oxazole, or imidazole derivatives.
One aspect of the invention includes COBRA compounds represented by the general formula I: 
where
X is O, S, or NH;
R is a saturated or unsaturated (C7 to C15) hydrocarbon chain;
R1 is hydrogen, halogen, OH, (C1 to C6) alkoxy, (C1 to C6) acyl, (C1 to C6) ester, or (C1 to C6) carboxylic acid;
Y is OH, SH, CN, halogen, or (C1 to C6) alkoxy; and
pharmaceutically acceptable salts thereof.
In another aspect of the invention, COBRA compounds are provided of the general formula II: 
where
X is NH, O, or S;
R is a saturated or unsaturated (C7 to C15) hydrocarbon chain;
R2 is hydrogen, OH, (C1 to C6) alkoxy, (C1 to C6) alkyl, (C1 to C6)alkenyl, (C1 to C6) alkynyl, (C3 to C7) cycloalkyl, aryl, heteroaryl, halogen, (C1 to C6) acyl, (C1 to C6) ester, or (C1 to C6) carboxylic acid;
Y is OH, SH, CN, halogen, or (C1 to C6) alkoxy; and
pharmaceutically acceptable salts thereof.
In yet another aspect of the invention, the COBRA compound has the general formula III: 
where
X is NH, O, or S;
R is a saturated or unsaturated (C7 to C15) hydrocarbon chain;
R2 is hydrogen, OH, (C1 to C6) alkoxy, (C1 to C6) alkyl, (C1 to C6)alkenyl, (C1 to C6) alkynyl, (C3 to C7) cycloalkyl, aryl, heteroaryl, halogen, (C1 to C6) acyl, (C1 to C6) ester, or (C1 to C6) carboxylic acid;
Y is OH, SH, CN, halo, or (C1 to C6) alkoxy;
and pharmaceutically acceptable salts thereof.
All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.
As used herein, xe2x80x9cacylxe2x80x9d refers to a group containing a carbon attached to oxygen by a double bond. Acyl groups can be a part of, for example, aldehydes or ketones. Typically, acyl groups include 1 to 15 carbon atoms or 1 to 6 carbon atoms. The carbon atoms can be aliphatic or aromatic. In some embodiments, the acyl group has 1 to 3 carbon atoms or 1 carbon atom.
The term xe2x80x9calkylxe2x80x9d refers to straight or branched hydrocarbon radicals, such as methyl, ethyl, propyl, butyl, pentyl, octyl, isopropyl, tert-butyl, sec-butyl, and the like. Typically, alkyl groups include 1 to 15 carbon atoms, 1 to 6 carbon atoms, or 1 to 3 carbon atoms.
As used herein, xe2x80x9calkythioxe2x80x9d comprises a sulfur attached to an alkyl by a single bond. Typically, alkythio groups include 1 to 15 carbon atoms, 1 to 6 carbon atoms, or 1 to 3 carbon atoms.
As used herein, xe2x80x9calkenexe2x80x9d and xe2x80x9calkenylxe2x80x9d, includes both branched and straight chain aliphatic hydrocarbon groups that have at least one double bond. Typically, alkene groups include 1 to 15 carbon atoms, 1 to 6 carbon atoms, or 1 to 3 carbon atoms.
The term xe2x80x9calkoxyxe2x80x9d refers to an oxygen atom substituted with an alkyl radical as defined above. Typical alkoxy groups include 1 to 15 or 1 to 6 carbon atoms or 1 to 3 carbon atoms such as methoxy, ethoxy, propoxy, iso-propoxy, and the like. Preferable alkoxy groups include methoxy and ethoxy.
As used herein, xe2x80x9calkynexe2x80x9d and xe2x80x9calkynylxe2x80x9d includes both branched and straight chain aliphatic hydrocarbon groups that have at least one triple bond. Typically, alkyne groups include 1 to 15 carbon atoms, 1 to 6 carbon atoms, or 1 to 3 carbon atoms.
As used herein xe2x80x9caminexe2x80x9d refers to compounds of the formula NRaRb and includes primary, secondary, and tertiary amines. Ra and Rb are each independently hydrogen, (C1 to C6) alkyl, aryl, heteroaryl, (C1 to C6) acyl, (C3 to C7) cycloalkyl, or Ra and Rb taken together with the nitrogen to which they are attached form a ring such as pyrrolidino, piperidino, morpholino, or thiomorpholino.
The term xe2x80x9carylxe2x80x9d refers to monovalent unsaturated aromatic carbocyclic radicals having a single ring, such as phenyl, or multiple fused rings, such as naphthyl or anthryl.
As used herein, xe2x80x9caryloxyxe2x80x9d comprises an oxygen attached to an aryl by a single bond.
As used herein, xe2x80x9ccycloalkylxe2x80x9d includes cyclic alkanes having 3 to 7 carbon atoms.
As used herein, an xe2x80x9cesterxe2x80x9d comprises a carbon attached to a first oxygen by a double bond and to a second oxygen by a single bond. The second oxygen is also attached to an alkyl group which has 1 to 15 carbon atoms, 1 to 6 carbon atoms, or 1 to 3 carbon atoms.
As used herein, the term xe2x80x9ccarboxylic acidxe2x80x9d comprises a carbon attached to a first oxygen by a double bond and to a second oxygen by a single bond. The second oxygen is also attached a hydrogen atom, i.e. COOH.
As used herein, the terms xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d refers to fluoride, chloride, bromide, and iodide radicals.
As used herein, xe2x80x9cheteroarylxe2x80x9d includes substituted or unsubstituted aromatic hydrocarbon compounds having at least one atom of O, N or S in an aromatic ring. Typical heteroaryl groups include, for example, furan, thiophene, pyrrole, thiazole, oxazole, or imidazole group. Heteroaryl groups can include two aromatic groups fused together such as, for example, benzothiophene, indole, carbazole, quinazoline, quinoline, and purine.
As used herein, the term xe2x80x9cthioacylxe2x80x9d refers to a group comprising a carbon atom attached to a sulfur atom by a double bond. Typically, the thioacyl group has 1 to 6 carbon atoms or 1 to 3 carbon atoms.
As used herein, xe2x80x9cpharmaceutically acceptable salt thereofxe2x80x9d includes an acid addition salt or a base salt.
As used herein, xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d includes any material which, when combined with a compound of the invention, allows the compound to retain biological activity, such as the ability to induce apoptosis of leukemia or breast tumor cells, and is non-reactive with the subject""s immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsions, and various types of wetting agents. Compositions comprising such carriers are formulated by well known conventional methods (see, for example, Remington""s Pharmaceutical Sciences, Chapter 43, 14th Ed., Mack Publishing Co., Easton, Pa.).
xe2x80x9cTreatingxe2x80x9d or xe2x80x9cTreatmentxe2x80x9d in the context of this invention means the prevention or reduction in severity of symptoms or effects of a pathological condition, including prolonging life expectancy. In the context of cancer therapy, treatment includes prevention of tumor growth, reduction of tumor size, enhanced tumor cell death, and increased apoptosis.
The COBRA binding pocket accommodates COBRA compounds. As used herein, the phrase xe2x80x9cCOBRA compoundxe2x80x9d refers to a compound having a non-linear polar group at one end of a molecule (the head) and a aliphatic, hydrophobic group at the other end of the molecule (the tail). The structure of the COBRA compounds increases the attraction of the compounds to the COBRA pocket and increases the residence time of the molecule in the binding pocket.
The residues of the COBRA binding pocket suitable for interaction with the tail part of COBRA compounds include Asp367, Leu217, Val275, Ile 276, Leu368, Tyr272, Ile212, Ile234, Gln233, Leu230, His229, Ile209, Ile231, and Leu23. Residues of the pocket suitable for interaction with the head part of COBRA compounds include Asn226, Pro222, and Ile219.
In general, the COBRA compounds of the invention include an aliphatic, hydrophobic tail part and a non-linear polar head part. The head part is a furan, thiophene, pyrrole, thiazole, oxazole, or imidazole group. The compounds are suitable for binding to the COBRA binding pocket of tubulin.
One aspect of the invention includes COBRA compounds represented by the general formula I: 
where
X is O, S, or NH;
R is a saturated or unsaturated (C7 to C15) hydrocarbon chain;
R1 is hydrogen, halogen, OH, (C1 to C6) alkoxy, (C1 to C6) acyl, (C1 to C6) ester, or (C1 to C6) carboxylic acid;
Y is OH, SH, CN, halogen, or (C1 to C6) alkoxy; and
pharmaceutically acceptable salts thereof.
The saturated or unsaturated (C7 to C15) hydrocarbon chain can be an alkyl, an alkenyl, or an alkynyl group. In some embodiments, the group is a C12 alkyl or a C12 alkylene. In one embodiment, R is C12H25.
The acyl, ester, carboxylic acid, or alkoxy groups can be substituted or unsubstituted. Suitable substituents include, for example, halogens, hydroxyl, amino, amino alkyl, acyl, thioacyl, CN, SH, ester, thioester, alkoxy, aryloxy, and alkylthio.
In some embodiments, R is C12H25; R1 is hydrogen, bromine, chlorine, CHO or COOH; Y is OH; and X is oxygen or sulfur. For example, the COBRA compounds of formula I can be: 
In another aspect of the invention, COBRA compounds are provided of the general formula II: 
where
X is NH, O, or S;
R is a saturated or unsaturated (C7 to C15) hydrocarbon chain;
R2 is hydrogen, OH, (C1 to C6) alkoxy, (C1 to C6) alkyl, (C1 to C6)alkenyl, (C1 to C6) alkynyl, (C3 to C7) cycloalkyl, aryl, heteroaryl, halogen, (C1 to C6) acyl, (C1 to C6) ester, or (C1 to C6) carboxylic acid;
Y is OH, SH, CN, halogen, or (C1 to C6) alkoxy; and
pharmaceutically acceptable salt thereof.
The saturated or unsaturated (C7 to C15) hydrocarbon chain can be an alkyl, an alkenyl, or an alkynyl group. In some embodiments, the group is a C12 alkyl or a C12 alkylene. In one embodiment, R is C12H25.
The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, acyl, ester, carboxylic acid, and alkoxy groups can be substituted or unsubstituted. Suitable substituents include, for example, halogens, hydroxyl, amino, amino alkyl, acyl, thioacyl, CN, SH, ester, thioester, alkoxy, aryloxy, and alkylthio.
In some embodiments of formula II, R is C12H25; Y is OH; X is S or NH; and R2 is hydrogen or a (C1 to C6) alkyl. For example, the COBRA compounds of formula II include 
In yet another aspect of the invention, the COBRA compound has the general formula III: 
where
X is NH, O, or S;
R is a saturated or unsaturated (C7 to C15) hydrocarbon chain;
R2 is hydrogen, OH, (C1 to C6) alkoxy, (C1 to C6) alkyl, (C1 to C6)alkenyl, (C1 to C6) alkynyl, (C3 to C7) cycloalkyl, aryl, heteroaryl, halogen, (C1 to C6) acyl, (C1 to C6) ester, or (C1 to C6) carboxylic acid;
Y is OH, SH, CN, halo, or (C1 to C6) alkoxy; and
pharmaceutically acceptable salt thereof.
The saturated or unsaturated (C7 to C15) hydrocarbon chain can be an alkyl, an alkenyl, or an alkynyl group. In some embodiments, the group is a C12 alkyl or a C12 alkylene. In one embodiment, R is C12H25.
The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, acyl, ester, carboxylic acid, and alkoxy groups can be substituted or unsubstituted. Suitable substituents include, for example, halogens, hydroxyl, amino, amino alkyl, acyl, thioacyl, CN, SH, ester, thioester, alkoxy, aryloxy, and alkylthio.
In some embodiments of formula III, R is C12H25; Y is OH; X is S or NH; and R2 is hydrogen or a (C1 to C6) alkyl. For example, the COBRA compounds of formula II include 
The compounds of the invention are capable of forming both pharmaceutically acceptable acid addition and/or base salts. Base salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Also included are heavy metal salts such as, for example, silver, zinc, cobalt, and cerium. Examples of suitable amines are N,Nxe2x80x2-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamene, N-methylglucamine, and procaine.
Pharmaceutically acceptable acid addition salts are formed with organic and inorganic acids. Examples of suitable acids for salt formation are hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, malic, gluconic, fumaric, succinic, ascorbic, maleic, methanesulfonic, and the like. The salts are prepared by contacting the free base form with a sufficient amount of the desired acid to produce either a mono or di, etc. salt in the conventional manner. The free base forms can be regenerated by treating the salt form with a base. For example, dilute solutions of aqueous base can be utilized. Dilute aqueous sodium hydroxide, potassium carbonate, ammonia, and sodium bicarbonate solutions are suitable for this purpose. The free base forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but the salts are otherwise equivalent to their respective free base forms for the purposes of the invention.
The compounds of the invention bind to tubulin at the COBRA binding pocket of tubulin. On binding of the tubulin binding compounds, tubulin is caused to depolymerize and/or inhibitition of tubulin assembly results. Suitable assays for the anti-tubulin acitivity of the inventive compounds are disclosed in the Examples below.
The compounds of the invention are useful to inhibit cell division and proliferation of non-cancerous cells. According to the method of the inveniton, disorders associated with cell proliferation are treated by administration of the compounds and compositions of the invention.
Such disorders include, for example, EBV-induced lymphoproliferative disease and lymphoma; neointimal hypoplasia, for example in patients with athlerosclerosis and patients undergoing balloon angioplasty; proliferative effects secondary to diabetes, including vascular proliferation and retinopathy; psoriasis; benign tumors, including angiomas, fiberomas, and myomas, histiocytosis, osteoporosis, mastocytosis, and myeleoproliferative disorders such as polycytemiavera.
The compounds of the invention are effective cytotoxic agents, for example, against tumor cells such as leukemia cells. In the methods of the invention, the cytotoxic effects are achieved by contacting cells, such as tumor cells, with micromolar amounts of the inhibitory compound.
The compounds of the invention can be used in methods of tumor treatment, for example, administering to a subject a compound of the invention in order to achieve an inhibition of tumor cell tubulin assembly and/or depolymerization of tumor cell tubulin, inhibition of tumor cell growth, a killing of tumor cells, induced apoptosis, and/or increased patient survival time.
The anti-cancer tubulin binding compounds of the invention are suitable for use in mammals. As used herein, xe2x80x9cmammalsxe2x80x9d means any class of higher vertebrates that nourish their young with milk secreted by mammary glands, including, for example, humans, rabbits, and monkeys.
The compounds of the present invention can be formulated as pharmaceutical compositions and administered to a mammalian host, including a human patient, in a variety of forms adapted to the chosen route of administration. The compounds are preferably administered in combination with a pharmaceutically acceptable carrier, and can be combined with or conjugated to specific delivery agents, including targeting antibodies and/or cytokines.
The compounds can be administered by known techniques, such as orally, parentally (including subcutaneous injection, intravenous, intramuscular, intrasternal or infusion techniques), by inhalation spray, topically, by absorption through a mucous membrane, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants or vehicles. Pharmaceutical compositions of the invention can be in the form of suspensions or tablets suitable for oral administration, nasal sprays, creams, sterile injectable preparations, such as sterile injectable aqueous or oleagenous suspensions or suppositories.
For oral administration as a suspension, the compositions can be prepared according to techniques well-known in the art of pharmaceutical formulation. The compositions can contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavoring agents. As immediate release tablets, the compositions can contain microcrystalline cellulose, starch, magnesium stearate and lactose or other excipients, binders, extenders, disintegrants, diluents, and lubricants known in the art.
For administration by inhalation or aerosol, the compositions can be prepared according to techniques well-known in the art of pharmaceutical formulation. The compositions can be prepared as solutions in saline, using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, or other solubilizing or dispersing agents known in the art.
For administration as injectable solutions or suspensions, the compositions can be formulated according to techniques well-known in the art, using suitable dispersing or wetting and suspending agents, such as sterile oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
For rectal administration as suppositories, the compositions can be prepared by mixing with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ambient temperatures, but liquefy or dissolve in the rectal cavity to release the drug.
Preferred administration routes include orally, parenterally, as well as intravenous, intramuscular or subcutaneous routes.
More preferably, the compounds of the present invention are administered parenterally, i.e., intravenously or intraperitoneally, by infusion or injection. In one embodiment of the invention, the compounds can be administered directly to a tumor by tumor injection. In another embodiment of the invention, the compounds can be administered using systemic delivery by intravenous injection.
Solutions or suspensions of the compounds can be prepared in water, isotonic saline (PBS), and optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene, glycols, DNA, vegetable oils, triacetin and mixtures thereof. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.
The pharmaceutical dosage form suitable for injection or infusion use can include sterile, aqueous solutions, dispersions, or sterile powders comprising an active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions. The final dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol such as glycerol, propylene glycol, or liquid polyethylene glycols, and the like, vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size, in the case of dispersion, or by the use of nontoxic surfactants. The prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion in the composition of agents delaying absorption such as, for example, aluminum monosterate hydrogels and gelatin.
Sterile injectable solutions are prepared by incorporating the conjugates in the required amount in the appropriate solvent with various other ingredients as enumerated above and, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
The compound of the invention can be targeted for specific delivery to the cells to be treated by conjugation of the compounds to a targeting moiety. Targeting moiety useful for conjugation to the compounds of the invention include antibodies, cytokines, and receptor ligands expressed on the cells to be treated.
The term xe2x80x9cconjugatexe2x80x9d refers to a complex formed with two or more compounds.
The phrase xe2x80x9ctargeting moietyxe2x80x9d refers to a compound which serves to deliver the compound of the invention to a specific site for the desired activity. Targeting moieties include, for example, molecules which specifically bind molecules present on a cell surface. Such targeting moieties useful in the invention include anti-cell surface antigen antibodies. Cytokines, including interleukins, factors such as epidermal growth factor (EGF), and the like, are also specific targeting moieties known to bind cells expressing high levels of their receptors.
Particularly useful targeting moieties for targeting the compounds of the invention to cells for therapeutic activity include those ligands that bind antigens or receptors present on the tumor cells to be treated. For example, antigens present on B-lineage cancer cells, such as CD19, can be targeted with anti-CD19 antibodies such as B43. Antibody fragments, including single chain fragments, can also be used. IL4 can also be used to target B-cells. Cancer cells expressing EGF or IGF receptors can be targeted with the binding ligand. Other such ligand-receptor binding pairs are known in the scientific literature for specific cancers. Methods for producing conjugates of the compounds of the invention and the targeting moieties are known.
When used in vivo to kill or inhibit the growth of tumor cells, the administered dose is that amount that can produce the desired effect, such as the amount sufficient to reduce or eliminate tumors. Appropriate amounts can be determined by those skilled in the art, extrapolating using known methods and relationships, from the in vitro data provided in the Examples.
In general, the dose of the COBRA tubulin binding compounds effective to achieve tumor cell apoptosis, reduction in tumors, and increased survival time, is 1-100 mg/kg body weight/dose for a direct targeted administration. The effective dose to be administered will vary with conditions specific to each patient. In general, factors such as the disease burden, tumor location (exposed or remote), host age, metabolism, sickness, prior exposure to drugs, and the like contribute to the expected effectiveness of a drug. One skilled in the art will use standard procedures and patient analysis to calculate the appropriate dose, extrapolating from the data provided in the Examples.
In general, a dose which delivers about 1-100 mg/kg body weight is expected to be effective, although more or less can be useful.
In addition, the compositions of the invention can be administered in combination with other anti-tumor therapies. In such combination therapy, the administered dose of the tubulin binding compounds can be less than for single drug therapy.
All publications, patents, and patent documents described herein are incorporated by reference as if fully set forth. The invention described herein can be modified to include alternative embodiments. All such obvious alternatives are within the spirit and scope of the invention, as claimed below.